Antigenic iron repressible protein from N. meningitidis related to the hemolysin family of toxins

ABSTRACT

An isolated, antigenic polypeptide comprises a segment having at least fifty amino acid residues. The amino acid sequence of the segment is present in N. meningitidis, and is different from, but substantially homologous with, the amino acid sequence of a segment of a member of the hemolysin family of toxins.

This application is continuation of Ser. No. 07/920,963 filed Jul. 28,1992 abandoned, which is a continuation-in-part of Ser. No. 07/552,649filed Jul. 16, 1990 abandoned, both of which are incorporated herein byreference.

The present invention is directed to antigenic polypeptides isolatedfrom Neisseria meningitidis, antibodies raised against the polypeptides,vaccines containing the polypeptides and DNA encoding the polypeptides.The polypeptides are members of the hemolysin family of toxins, atypical member of which is alpha-hemolysin from E. coli.

Bacterial pathogenesis is a complicated and often poorly understoodprocess. Many pathogenic bacteria secrete toxins that impair themetabolism and function of animal cells. Various classes of moleculesconstitute such toxins.

An example of a protein toxin is found in pathogenic E. coli strainsthat cause extra-intestinal infections in humans. Such infections arecharacterized by the lysis of mammalian erythrocytes. The hemolyticactivity is due to a class of toxins known as hemolysin. The classincludes alpha-hemolysin and beta-hemolysin; see Welch et al, Infectionand Immunity 42, 178-186 (1983).

Another protein toxin is adenylate cyclase, which is found in Bordetellapertussis and Bacillus anthracis, and which impairs functions ofprofessional phagocytes. These bacteria are responsible for whoopingcough and anthrax, respectively.

A third class of protein toxins from pathogenic bacteria are theleukotoxins, which are found in Actinobacillus actinomycetemcomitans,which is the etiologic agent of localized juvenile periodontitis, andPasteurella haemolytica, which kills bovine leukocytes.

Interestingly, the adenylate cyclase from B. pertussis and B. anthracisand the leukotoxins from A. actinomycetemcomitans and P. haemolyticahave amino acid sequences that exhibit considerable homology with thatof alpha-hemolysin from E. Coli; see Glaser et al, Molecular Biology 2,19-30 (1988) and Kolodrubetz et al, Infection and Immunity 57, 1465-1469(1989). Apparently, there is a class of toxins found in various generaof bacteria. The amino acid sequence of this family of cytotoxins ischaracterized by a highly repeated nine amino acid motif, LxGGxGNDx,wherein x represents any amino acid. For the purposes of thisspecification, this family of toxins will be referred to as thehemolysin family of toxins.

It should be understood that "hemolysin family of toxins" is a genericname familiar to those in the art, and is not meant to imply that allmembers are hemolytic, or, for that matter, cytotoxic, although mostare. Membership in the family depends on the existence of homology inthe amino acid sequence, as defined below. In addition to thosementioned above, homologous proteins have also been found in Serratiaand Proteus, although it is not certain whether these members of thehemolysin family are, in fact, cytotoxic.

Little is known about the intriguing and sometimes fatal bacteriaNeisseria meningitidis, which is responsible for spinal meningitis andseptic shock. N. meningitidis and the diseases it causes have beenreviewed by Paterson in "Neisseria meningitidis and MeningococcalDisease" in Biologic and Clinical Basis of Infectious Diseases, W. B.Saunders Company, Chapter 43 (1980).

The genotype of N. meningitidis is very similar to that of N.gonorrhoeae, although the phenotype is quite different. It is oftenimportant to distinguish between these Neisseria species. Immunologicspeciation is often difficult due to a lack of sufficient amounts ofgroup-specific antigens.

N. meningitidis exists as various serotypes, the prevalence of whichvaries with time and location. The serotypes include A, B, C, D, X, Y,Z, 29-E and W-135.

The three most important known antigenic and/or toxic constituents of N.meningitidis infections are a capsular polysaccharide, alipopolysaccharide-endotoxin cell wall complex and a Neisseria-specificprotein. The capsular polysaccharide is a major virulence factor thatenables meningococci to resist phagocytosis by segmented neutrophils.

Vaccines containing meningococcal polysaccharides are used against someof the serotypes of N. meningitidis. For example, protection against theA, C, Y and W-135 serotypes is afforded by polysaccharide vaccines. Suchvaccines are, however, inadequate for general protection againstinfection by N. meningitidis. For example, the immune response ofserotypes A and C to polysaccharide vaccines is poor, especially inchildren under two years old, who constitute the group most susceptibleto meningococcal disease. Moreover, no effective vaccine exists forserotype B, possibly because the group B capsular polysaccharide isrelatively non-immunogenic.

It is apparent that much needs to be learned about the pathology of N.meningitidis. Possibly, additional understanding of this pathogen willlead to the discovery of useful vaccines in general for more serotypesthan are currently available.

For example, it is not known why the colonization of the respiratorytract by N. meningitidis progresses to acute meningococcal disease andsometimes death in an occasional individual, whereas it does not do soin the great majority of others who are apparently at comparable risk.The amount of neither capsular polysaccharide norlipopolysaccharide-endotoxin complex correlates with the seriousness ofthis disease. Exposure to microorganisms with antigenic constituentsthat cross-react with capsular polysaccharides of N. meninaitidis hasbeen proposed as an explanation; see Paterson, id.

Other explanations are also possible. For example, cross-immunity toantigens other than capsular polysaccharides cannot be ruled out. It isinteresting to note in this regard that there are no known proteintoxins associated with N. meningitidis. One reason for this may be thatN. meningitidis is often cultured in vitro under iron-rich conditionsthat do not exist in a human host. It is known, however, that somemeningococcal proteins are iron-repressed and are not observed in vitro,although they are expressed in vivo. See Black et al., Infection andImmunity 54, 710-713 (1986) and Brener et al, ibid. 33, 59-66 (1981).

One problem addressed by the present invention is the discovery ofantigenic polypeptides and DNA sequences that are capable of identifyingN. meningitidis and distinguishing it from N. gonorrhoeae. Anotherproblem addressed by the present invention is the discovery of proteinscapable of producing antibodies effective against meningococcal disease.

SUMMARY OF THE INVENTION

These and other problems as will be apparent to those having ordinaryskill in the art have been solved by providing an isolated, antigenicpolypeptide comprising a segment having at least fifty amino acidresidues, wherein the amino acid sequence of the segment is present inN. meninaitidis, and wherein the amino acid sequence is different from,but substantially homologous with, the amino acid sequence of a segmentof a member of the hemolysin family of toxins.

Another way of defining the polypeptide is to say that it is an isolatedpolypeptide comprising a segment having an amino acid sequence presentin N. meningitidis wherein the amino acid sequence consists of at leastthree repeats of the nine amino acid hemolysin consensus sequence, thehemolysin consensus sequence consisting of at least four of:

L at position 1;

G at position 3;

G at position 4;

G at position 6;

N at position 7;

D at position 8; and

X at positions 2, 5 and 9;

wherein x, independently, represents any single amino acid residue.

The invention further includes antigenic fragments of such polypeptides,antibodies raised against such polypeptides, nucleotide sequencesencoding such polypeptides, and vaccines containing such polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the relationship to one another of the restrictionfragments used to obtain the frpA gene.

FIGS. 2A-2G show the DNA and amino acid sequence of the gene for anantigenic iron repressible protein (frpA) from N. meningitidis relatedto the hemolysin family of toxins. See Seq. ID No. 1.

FIGS. 3A-3E show the putative amino acid sequence for the DNA sequenceof FIGS. 2A-2G. See Seq. ID No. 2.

DETAILED DESCRIPTION OF THE INVENTION The Polypeptide Segment

It has unexpectedly been discovered that when N. meningitidis is grownunder iron-limiting conditions, a polypeptide comprising a segmenthaving an amino acid sequence that is different from, but substantiallyhomologous with, a segment of the hemolysin family of toxins isexpressed. Monoclonal antibodies raised against the polypeptide found inN. meningitidis, such as A4.85 (see below), cross-react in Western blotswith alpha-hemolysin (HlyA) produced by the hlyA gene in E. coli andadenylate cyclase produced in Bordetella pertussis.

The hemolysin family of toxins, as used herein, includes the homologous,cytotoxic or proteolytic polypeptides found in bacteria of the generaEscherichia, Serratia, Pasteurella, Proteus, Actinobacillus, andBordetella. The family specifically includes alpha-hemolysin,leukotoxin, and adenylate cyclase.

Determinations whether two amino acid sequences are substantiallyhomologous are, for the purpose of the present specification, based onFASTA searches in accordance with Pearson and Lipman, Proc. Natl. Acad.Sci. USA 85, 2444-2448 (1988). A substantially homologous sequence inaccordance with the present invention has at least 15% identity,preferably at least 20% identity and, more preferably, at least 25%identity in amino acid sequence when determined in accordance with themethod of Pearson and Lipman, which is incorporated herein by reference.

The polypeptide of the present invention need not contain a segment thatis identical to other members of the hemolysin family of toxins. Theidentity in accordance with the FASTA method may be as high as 90% or95%, but when isolated from N. meningitidis normally does not exhibitidentities greater than 40% or 50%.

The size of the polypeptide is not critical, as long as it contains asegment that is substantially homologous to a segment of a member of thehemolysin family of toxins. The substantially homologous segment has atleast fifty amino acid residues, preferably at least 100 amino acidresidues, and more preferably at least 200 amino acid residues. In thisspecification, the word "polypeptide" will be consideredindistinguishable from words like "protein" and "peptide."

The segment of the meningococcal polypeptide that is substantiallyhomologous to a segment of the hemolysin family of toxins contains thesame nine amino acid motif that is characteristic of all the members ofthe hemolysin family of toxins. The consensus sequence is LxGGxGNDx,hereinafter hemolysin consensus sequence. The amino acid represented byx may be any single amino acid.

The polypeptide of the invention may be defined in terms of thehemolysin consensus sequence as well as by the substantial homologystandard described above. For this purpose, a nine amino acid sequenceis considered to be a hemolysin consensus sequence if it contains atleast four, preferably at least five, and more preferably all six of thespecifically defined amino acid residues (i.e. L-GG-GND-) at the correctposition.

Referring to FIGS. 3A-3E, which, as described below, is a polypeptideisolated from N. meninaitidis, the homologous segment comprises twostretches of multiple repeats of the hemolysin consensus sequence, i.e.between amino acids 763 and 789; and between amino acids 900 and 989.There are 13 complete consensus sequences in FIGS. 3A-3E. See Seq. IDNo. 2.

The polypeptides of the present invention contain segments that arepresent in N. meningitidis and that comprise at least three, preferablyat least five, and more preferably at least ten hemolysin consensussequences. The polypeptides of the invention may have as many as atleast 21 hemolysin consensus sequences.

The polypeptide is isolated, which means that it is essentially free ofother proteins, especially of other proteins from N. meningitidis.Essentially free from other proteins means that it is at least 90%,preferably at least 95% and, more preferably, at least 98% free of otherproteins.

Preferably, the polypeptide is essentially pure, which means that thepolypeptide is free not only of other polypeptides, but also of othermaterials used in the isolation and identification of the polypeptide,such as, for example, sodium dodecyl sulfate and other detergents aswell as nitrocellulose paper. The polypeptide is at least 90% free,preferably at least 95% free and, more preferably, at least 98% free ofsuch materials.

The polypeptide of the present invention is antigenic, which means thatthe polypeptide induces specific antibodies in a mammal. Preferably, thepolypeptide is immunogenic.

The polypeptide may be the entire polypeptide as it exists in N.meningitidis, or an antigenic, preferably immunogenic, fragment of thewhole polypeptide. Antigenic and/or immunogenic fragments of antigenicand/or immunogenic polypeptides may be identified by methods known inthe art. Usually, the antigenic fragment will comprise at least aportion of the segment having an amino acid sequence that is differentfrom, but homologous to, the amino acid sequence of a segment of apolypeptide that is a member of the hemolysin family of toxins, or willcomprise at least a portion of the segment having at least three,preferably at least five, and more preferably at least ten hemolysinconsensus sequences.

Preparation of the Polypeptide

The polypeptides of the present invention may be prepared by methodsknown in the art. Such methods include isolating the polypeptidedirectly from N. meninaitidis; isolating or synthesizing DNA encodingthe polypeptide and using the DNA to produce recombinant polypeptide;and synthesizing the polypeptide from individual amino acids.

The polypeptide or DNA encoding the polypeptide may be isolated from anyserotype of N. meningitidis. Such serotypes include A, B, C, D, X, Y, Z,29-E and W-135.

Suitable sources of meningococcal strains from which the polypeptide andDNA encoding the polypeptide may be isolated are available. Such sourcesinclude the American Type Culture Collection (Bethesda, Md.) and theNeisseria Repository (NAMRU, University of California, Berkeley).Suitable strains include FAM18 and FAM20 (Dyer et al, MicrobialPathogenesis 3, 351-363 (1987)), and FAM 19. Additional meningococcalstrains are described by Schryvers and Morris in Infection and Immunity56, 1144-1149 (1988).

The polypeptide may be isolated directly from N. meningitidis by methodsknown in the art. First, meningococcal outer membranes are isolated andprepared by known methods. The methods described by West and Sparling inInfect. Immun. 47, 388-394 (1985) and by Schryvers and Morris in Infect.Immun. 56, 1144-1149 (1988) are suitable.

The isolated membrane proteins may be solubilized by known methods, suchas the addition of detergents. Commonly used detergents includeOctyl-B-Glucoside, Chaps, Zwittergent 3.14 or Triton-X. The use ofdetergents to enhance solubility of membrane proteins is described byJones et al. in Finby, Solubilization and Reconstitution of MembraneProteins: A Practical Approach, IRL Press (1986), Helenius et al. inBiochim. Biophys. Acta 415, 29 (1975) and Hjelmeland and Chrambach,Methods Enzymol. 104, 305 (1984).

Proteins are isolated from the solubilized membrane fraction by standardmethods. Some suitable methods include precipitation and liquidchromatographic protocols such as ion exchange, hydrophobic interactionand gel filtration. See, for example, Methods Enzymol. 182 (Guide toProtein Chemistry, Deutscher, Ed. Section VII) 309 (1990) and Scopes,Protein Purification. Springer-Verlag, New York (1987).

Alternatively, purified material is obtained by separating the proteinon preparative SDS-PAGE gels, slicing out the band of interest andelectroeluting the protein from the polyacrylamide matrix by methodsknown in the art. The detergent SDS is removed from the protein by knownmethods, such as by dialysis or the use of a suitable column, such asthe Extracti-Gel column from Pierce.

The polypeptide may also be produced by isolating DNA that encodes thepolypeptide; cloning the DNA in a suitable host; expressing the DNA inthe host; and harvesting the polypeptide.

The first DNA encoding the polypeptide of the present invention wasisolated by an immunoscreening method. Such methods are described byManiatis et al in "Molecular Cloning: A Laboratory Manual," Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1982).

Briefly, monoclonal antibodies were generated against iron-stressedouter membrane proteins of N. meningitidis strain FAM20. One monoclonalantibody, A4.85, recognized several iron-regulated proteins in Westernblots of the FAM20 outer membranes. A4.85 was used to isolate a clonefrom an FAM20 genomic library constructed in the expression vectorlambda-gt11. The sequence of this clone was determined and used to cloneadjacent genomic restriction fragments. The adjoined DNA sequence ofthis region contained a long open reading frame, which is included inFIGS. 2A-G. See Seq. ID No. 1. The amino acid sequence predicted fromthe nucleotide sequence of FIGS. 2A-G is shown as FIGS. 3A-E. See Seq.ID No. 2.

A FASTA homology search in accordance with Pearson and Lipman, Proc.Natl. Acad. Sci. USA 85, 2444-2448 (1988) of the amino acid sequencededuced from the open reading frame (FIGS. 3A-E) was performed.Surprisingly, the amino acid sequence exhibited substantial homology toseveral members of the hemolysin family of toxins, as discussed above.

As further evidence that the polypeptide isolated from N. meningitidisis a member of the hemolysin family of toxins, the antibody raisedagainst, and used to isolate, the polypeptide shown as FIGS. 3A-E (SeeSeq. ID No. 2.), A4.85, cross-reacted strongly with alpha-hemolysin(HlyA) from E. coli and with adenylate cyclase produced in Bordetellapertussis.

The immunoscreening method may be repeated in order to obtain additionalfragments of the gene encoding the polypeptide of the invention or toobtain the gene encoding the entire polypeptide. It is, of course, notnecessary to repeat the immunoscreening process. The entire gene oradditional fragments of the gene are preferably isolated by using theknown DNA sequence or fragments thereof as a probe. To do so,meningococcal DNA restriction fragments, either flanking the ends of theregion already cloned or containing the entire region, are identified bySouthern hybridization using labelled oligonucleotide probes derivedfrom a previously determined sequence, such as that shown as FIGS. 2A-G,or a fragment thereof. See Seq. ID No. 1.

The DNA obtained may be amplified by methods known in the art. Onesuitable method is the polymerase chain reaction (PCR) method describedby Mullis et al in U.S. Pat. No. 4,683,195 and by Sambrook, Fritsch andManiatis (eds) in Molecular Cloning, A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1989). It is convenient toamplify the clones in the lambda-gt11 vectors using lambda-gt11-specificoligomers as the amplimers (available from New England Biolabs, Beverly,Ma.).

The restriction fragments are cloned into a suitable vector, such as aplasmid or bacteriophage, and sequenced in accordance with methods knownin the art. A suitable sequencing method is the dideoxy chainterminating method described by Sanger et al in Proc. Natl. Acad. Sci.USA 74, 5463-5467 (1977). Suitable vectors and polymerases forsequencing are known. A suitable vector is the lambda-gt11 specificprimer. (New England Biolabs, Beverly, Ma.). A suitable polymerase isSequenase (United States Biochemical Corp., Cleveland, Ohio).

The DNA encoding the polypeptide of the invention may be used to expressrecombinant polypeptide in a wide variety of host cells using a widevariety of vectors. The host may be prokaryotic or eukaryotic. The DNAmay be obtained from natural sources and, optionally, modified. Thegenes may also be synthesized in whole or in part.

Cloning vectors may comprise segments of chromosomal, non-chromosomaland synthetic DNA sequences. Some suitable prokaryotic vectors includeplasmids from E. coli, such as colE1, pCR1, DBR322, pMB9, and RP4.Prokaryotic vectors also include derivatives of phage DNA such as M13,f1, and other filamentous single-stranded DNA phages.

Vectors for expressing proteins in bacteria, especially E.coli, are alsoknown. Such vectors include pK233 (or any of the tac family ofplasmids), T7, and lambda P_(L). Examples of vectors that express fusionproteins include the PATH vectors described by Dieckmann and Tzagoloffin J. Biol. Chem. 260, 1513-1520 (1985). These vectors contain DNAsequences that encode anthranilate synthetase (TrpE) followed by apolylinker at the carboxy terminus. Other expression vector systems arebased on beta-galactosidase (pEX); maltose binding protein (pMAL); andglutathione S-transferase (pGST)--see Gene 67, 31 (1988) and PeptideResearch 3, 167 (1990).

Vectors useful in yeast are available. A suitable example is the 2μplasmid.

Suitable vectors for use in mammalian cells are also known. Such vectorsinclude well-known derivatives of SV-40, adenovirus, retrovirus-derivedDNA sequences and vectors derived from combination of plasmids and phageDNA.

Further eukaryotic expression vectors are known in the art (e.g., P. J.Southern and P. Berg, J. Mol. Appl. Genet. 1, 327-341 (1982); S.Subramani et al, Mol. Cell. Biol. 1, 854-864 (1981); R. J. Kaufmann andP. A. Sharp, "Amplification And Expression Of Sequences Cotransfectedwith A Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol.Biol. 159, 601-621 (1982); R. J. Kaufmann and P. A. Sharp, Mol. Cell.Biol. 159, 601-664 (1982); S. I. Scahill et al, "Expression AndCharacterization Of The Product Of A Human Immune Interferon DNA Gene InChinese Hamster Ovary Cells," Proc. Natl. Acad. Sci. USA 80, 4654-4659(1983); G. Urlaub and L. A. Chasin, Proc. Natl. Acad. Sci. USA 77,4216-4220, (1980).

Useful expression hosts include well-known prokaryotic and eukaryoticcells. Some suitable prokaryotic hosts include, for example, E. coli,such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coli X1776, E.coli X2282, E. coli DHI, and E. coli MRCl, Pseudomonas, Bacillus, suchas Bacillus subtilis, and Streptomyces. Suitable eukaryotic cellsinclude yeasts and other fungi, insect, animal cells, such as COS cellsand CHO cells, human cells and plant cells in tissue culture.

The expression vectors useful in the present invention contain at leastone expression control sequence that is operatively linked to the DNAsequence or fragment to be expressed. The control sequence is insertedin the vector in order to control and to regulate the expression of thecloned DNA sequence. Examples of useful expression control sequences arethe lac system, the try system, the tac system, the trc system, majoroperator and promoter regions of phage lambda, the control region of f1coat protein, the glycolytic promoters of yeast, e.g., the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,e.g., Pho5, the promoters of the yeast alpha-mating factors, andpromoters derived from polyoma, adenovirus, retrovirus, and simianvirus, e.g., the early and late promoters or SV40, and other sequencesknown to control the expression of genes of prokaryotic or eukaryoticcells and their viruses or combinations thereof.

The recombinant polypeptide is purified by methods known in the art.Suitable methods are described F. A. O. Marston, "The Purification ofEukaryotic Polypeptides Expressed in Escherichia coli," in DNA Cloning,D. M. Glover, Ed., Vol. III, IRL Press Limited, England (1987).

The polypeptide of the invention and DNA encoding the polypeptide mayalso be chemically synthesized from individual amino acid residues andnucleotides, respectively, by methods known in the art. Suitable methodsfor synthesizing the polypeptide are described by Stuart and Young in"Solid Phase Peptide Synthesis," Second Edition, Pierce Chemical Company(1984). Suitable methods for synthesizing DNA are described by Caruthersin Science 230, 281-285 (1985).

Vaccines

A polypeptide comprising a segment having an amino acid sequence that isdifferent from, but substantially homologous with, the amino acidsequence of a member of the hemolysin family of toxins is, unexpectedly,an antigen useful for protecting a mammal from infectious diseasescaused by N. Meningitidis. The mammal is typically a human.

To be useful, the antigen is non-toxic to the mammal being immunized. Ifthe antigen is toxic, it may be detoxified by methods known in the art.Such methods include, for example, providing antigenic, non-toxicfragments of the entire polypeptide or detoxifying a polypeptide by, forexample, binding the toxin to a carrier molecule that destroys toxicity,but does not affect antigenicity. The carrier molecule is typicallyanother polypeptide.

Preferably, an amino acid sequence of the antigen is present in apolypeptide found in N. meningitidis. The polypeptide or non-toxic,antigenic fragments useful in immunizing mammals may be made by methodsknown in the art, such as by isolation from N. meningitidis, productionby recombinant DNA techniques, or chemical synthesis, as describedabove.

The length of the fragment is not critical as long as the fragment isantigenic and non-toxic. Therefore, the fragment should containsufficient amino acid residues to define the epitope. Methods forisolating and identifying antigenic fragments from known antigenicpolypeptides are described by Salfeld et al. in J. Virol. 63, 798-808(1989) and by Isola et al. in J. Virol. 63, 2325-2334 (1989).

If the fragment defines the epitope, but is too short to be antigenic,it may be conjugated to a carrier molecule. Some suitable carriermolecules include keyhole limpet hemocyanin and bovine serum albumen.Conjugation may be carried out by methods known in the art. One suchmethod is to combine a cysteine residue of the fragment with a cysteineresidue on the carrier molecule.

The present invention further includes vaccine compositions forimmunizing mammals, including humans, against infection by N.meningitidis. The vaccine comprises an immunogenic antigen as describedabove in a suitable carrier. Suitable carriers include any of thestandard pharmaceutically acceptable carriers, such as water, phosphatebuffered saline solution, and emulsions.

The vaccine may include adjuvants, such as muramyl peptides, andlymphokines, such as interferon, interleukin-1 and interleukin-6. Theantigen may be adsorbed on suitable particles, such as aluminum oxideparticles, or encapsulated in liposomes, as is known in the art.

The invention further includes methods of immunizing host mammals,including humans, by administering the vaccine compositions describedabove to mammals in need of protection from diseases caused by N.meningitidis. The vaccine comprises an immunogenic polypeptide in a formthat is non-toxic to mammals. The polypeptide comprises an amino acidsequence that is homologous with the amino acid sequence of a member ofthe hemolysin family of toxins. The amino acid sequence is preferablypresent in N. meningitidis, and is usually found in the outer membranesof N. meningitidis. Since, however, antibodies cross-react with thepolypeptide of the invention and members of the hemolysin family oftoxins from other genera of bacteria, the antigen in the vaccinecomposition may comprise an amino acid sequence in such other genera,such as from E. coli or B. pertussis.

The vaccine may be administered to a mammal by methods known in the art.Such methods include, for example, intravenous, intraperitoneal,subcutaneous, or intramuscular administration.

Antibodies

The present invention provides antibodies raised against a polypeptideof the invention. The polypeptide comprises an amino acid sequence thatdefines an epitope, and is substantially homologous with the amino acidsequence of a member of the hemolysin family of toxins. The antibodiesare preferably raised against a polypeptide comprising an amino acidsequence that is present in N. Meningitidis, and that is different frompolypeptides that are members of the hemolysin family of toxins fromother genera of bacteria.

The antibodies are preferably monoclonal. Monoclonal antibodies may beproduced by methods known in the art. These methods include theimmunological method described by Kohler and Milstein in Nature 256,495-497 (1975) and the recombinant DNA method described by Huse et al inScience 246, 1275-1281 (1989).

Mammals, including humans, suffering from diseases caused by infectionwith N. meningitidis may be treated by administering antibodies specificto a member of the hemolysin family of toxins. Antibodies raised againsta member of the hemolysin family of toxins from any genera of bacteriaare suitable, although antibodies raised against a polypeptidecomprising an amino acid sequence present in N. meningitidis ispreferred.

For therapeutic purposes, it is necessary for the antigenic polypeptidesof the invention to produce neutralizing antibodies. Neutralizingantibodies are antibodies that significantly inhibit the growth of orkill the bacterial cells and/or significantly neutralize the toxinfunction of the polypeptide in vitro or in vivo. Growth of the bacteriais significantly inhibited or the toxin function of the polypeptide issignificantly neutralized in vivo if the inhibition or neutralization issufficient to prevent or reduce the symptoms of the disease of a mammalinfected with the disease.

Neutralizing antibodies may also be used to produce anti-idiotypicantibodies useful as vaccines for immunizing mammals, including humans,suffering from diseases caused by infection with N. meningitidis.Anti-idiotypic antibodies are prepared in accordance with methods knownin the art.

Nucleic Acid Molecules

The present invention also includes isolated nucleic acid molecules thatencode any of the polypeptides of the invention described above. Thenucleic acid molecule may be DNA or RNA.

The utility of the nucleic acid molecule lies in its ability to be usedas a probe for detecting N. meningitidis, as explained below, or toproduce a polypeptide of the invention, as explained above. The nucleicacid molecule may be prepared by methods known in the art. Suitablemethods include isolating the DNA from N. Meninaitidis or synthesizingthe DNA in accordance with known procedures as described above.

Probes

The present invention further provides a method of detecting thepresence of N. meningitidis in a sample. The method involves use of aprobe that recognizes a polypeptide that is a member of the hemolysinfamily of toxins, and, in particular, a member of the hemolysin familyof toxins present in N. meningitidis, or a gene encoding such apolypeptide. The probe recognizes N. meningitidis if present in thesample.

The probe may be an antibody, preferably a monoclonal antibody. Theantibodies may be prepared as described above.

Methods are known for detecting polypeptides with antibodies. Forexample, a polypeptide may be immobilized on a solid support.Immobilization of the polypeptide may occur through an immobilized firstantibody specific for the polypeptide. The immobilized first antibody isincubated with a sample suspected of containing the polypeptide. Ifpresent, the polypeptide binds to the first antibody.

A second antibody, also specific for the polypeptide, binds to theimmobilized polypeptide. The second antibody may be labelled by methodsknown in the art. Non-immobilized materials are washed away, and thepresence of immobilized label indicates the presence of the polypeptide.This and other immunoassays are described by David, et al., in U.S. Pat.No. 4,376,110 assigned to Hybritech, Inc., La Jolla, Calif.

The probe may also be a nucleic acid molecule that recognizes a RNA orDNA molecule that encodes a member of the hemolysin family of toxinspresent in N. meningitidis. Methods for determining whether a nucleicacid molecule probe recognizes a specific nucleic acid molecule in asample are known in the art. Generally, a labelled probe that iscomplementary to a nucleic acid sequence suspected of being in a sampleis prepared. The presence of probe hybridized to the target nucleic acidmolecule indicates the presence of the nucleic acid molecule. Suitablemethods are described by Schneider et al in U.S. Pat. No. 4,882,269,which is assigned to Princeton University, and by Segev in PCTApplication WO 90/01069. The Schneider et al patent and the Segevapplication are both licensed to ImClone Systems Inc., New York City.

The probes described above are labelled in accordance with methods knownin the art. Methods for labelling antibodies have been described, forexample, by Hunter and Greenwood in Nature 144, 945 (1962) and by Davidet al in Biochemistry 13, 1014-1021 (1974). Additional methods forlabelling antibodies have been described in U.S. Pat. Nos. 3,940,475 and3,645,090. Methods for labelling oligonucleotide probes have beendescribed, for example, by Leary et al, Proc. Natl. Acad. Sci. USA(1983) 80:4045; Renz and Kurz, Nucl. Acids Res. (1984) 12:3435;Richardson and Gumport, Nucl. Acids Res. (1983) 11:6167; Smith et al,Nucl. Acids Res. (1985) 13:2399; and Meinkoth and Wahl, Anal. Biochem.(1984) 138:267.

The label may be radioactive. Some examples of useful radioactive labelsinclude ³² P, ¹²⁵ I, ¹³¹ I, and ³ H. Use of radioactive labels have beendescribed in U.K. 2,034,323, U.S. Pat. No. 4,358,535, and U.S. Pat. No.4,302,204.

Some examples of non-radioactive labels include enzymes, chromophors,atoms and molecules detectable by electron microscopy, and metal ionsdetectable by their magnetic properties.

Some useful enzymatic labels include enzymes that cause a detectablechange in a substrate. Some useful enzymes and their substrates include,for example, horseradish peroxidase (pyrogallol and o-phenylenediamine),beta-galactosidase (fluorescein beta-D-galactopyranoside), and alkalinephosphatase (5-bromo-4-chloro-3-indolyl phosphate/nitro bluetetrazolium). The use of enzymatic labels have been described in U.K.2,019,404, EP 63,879, and by Rotman, Proc. Natl. Acad. Sci., 47,1981-1991 (1961).

Useful chromophores include, for example, fluorescent, chemiluminescent,and bioluminescent molecules, as well as dyes. Some specificchromophores useful in the present invention include, for example,fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone,luminol.

The labels may be conjugated to the antibody or nucleotide probe bymethods that are well known in the art. The labels may be directlyattached through a functional group on the probe. The probe eithercontains or can be caused to contain such a functional group. Someexamples of suitable functional groups include, for example, amino,carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate.

The label may also be conjugated to the probe by means of a ligandattached to the probe by a method described above and a receptor forthat ligand attached to the label. Any of the known ligand-receptorcombinations is suitable. The biotin-avidin combination is preferred.

The polypeptide of the invention may be used to detect the presence ofantibodies specific for N. meningitidis in a sample. The methodcomprises preparing a polypeptide containing a segment having an aminoacid sequence that is substantially homologous to a member of thehemolysin family of toxins. The polypeptide may be prepared as describedabove. Preferably, the polypeptide comprises a segment having an aminoacid sequence that is present in N. meningitidis.

The sample may, for example, be from a patient suspected of beinginfected with N. meningitidis. Suitable assays are known in the art,such as the standard ELISA protocol described by R. H. Kenneth,"Enzyme-Linked Antibody Assay with Cells Attached to Polyvinyl ChloridePlates" in Kenneth et al, Monoclonal Antibodies, Plenum Press, N.Y.,page 376 (1981).

Briefly, plates are coated with antigenic polypeptide at a concentrationsufficient to bind detectable amounts of the antibody. After incubatingthe plates with the polypeptide, the plates are blocked with a suitableblocking agent, such as, for example, 10% normal goat serum. The sample,such as patient sera, is added and titered to determine the endpoint.Positive and negative controls are added simultaneously to quantitatethe amount of relevant antibody present in the unknown samples.Following incubation, the samples are probed with goat anti-human Igconjugated to a suitable enzyme. The presence of anti-polypeptideantibodies in the sample is indicated by the presence of the enzyme.

Antibodies raised against polypeptides of the present invention arecapable of recognizing N. meningitidis and distinguishing meningococcalcells from gonococcal cells in a sample. The A4.85 monoclonal antibody,for example, recognizes a polypeptide expressed by iron-stressedmeningococcal cells. A4.85 does not, however, recognize any proteins iniron-stressed N. gonorrhoeae.

The antibodies may be labelled by known methods as described above.Assays for distinguishing iron-stressed meningococcal cells fromiron-stressed gonococcal cells follow known formats, such as standardblot and ELISA formats.

EXAMPLES Example 1

Isolation of A4.85 MAb

Bacterial outer membranes are prepared from iron-stressed cultures ofNeisseria meningitidis strain FAM20 as follows. FAM20 is inoculated intochelexed defined medium (CDM, West et al, J. Bacteriology 169, 3414(1987)). This medium allows growth only until iron stores within thebacteria have been depleted. During this time, a set of proteins thatare regulated by the availability of iron become expressed. Bacteria areharvested and outer membranes are prepared as described by Dyer et al inInfection and Immunity 56, 977 (1988).

Three to five BALB/c female mice are immunized with iron-stressed FAM20outer membranes by either the intramuscular (im) or intraperitoneal (ip)routes. With the im route, 100 μg of antigen (Ag) is emulsified incomplete Freund's adjuvant and injected on two different sites on dayzero, followed by booster doses two weeks apart with the Ag nowemulsified in incomplete Freund's adjuvant. The ip route involvesimmunization with 100 μg of Ag on days zero, 7, 14 and 28. Serumantibody levels are checked by either ELISA or Western blotting threedays following the final boost to determine serum antibody levels. Miceare given a final boost ip on each of three consecutive days before thefusion. On the day of the fusion, mice are sacrificed by cervicaldislocation and the spleens are removed aseptically. Spleen cells areextracted by teasing the cells out of the sac using two bent 19 ganeedles. Extracted cells are resuspended to give single cellsuspensions.

Mouse myeloma cells, SP2.0-AG14 (ATCC CRL 1581), that have been grown inDulbecco's Modified Eagle's Medium/Ham's F-12 (DMEM/F12) supplementedwith 15% fetal calf serum (FCS), are used as the fusion partner. Cellsare mixed in a 10:1 ratio of spleen:myeloma cells and pelleted togetherin a 50 ml centrifuge tube. The supernatant is aspirated off leaving adry pellet to which 1 ml of 50% polyethylene glycol (PEG) (prewarmed to37° C.) is added. The cells are gently resuspended and allowed to sit atroom temperature for 2 minutes, after which 1 ml of DMEM/F12 withoutadded sera is added and the cells gently resuspended. The cells are thenfurther diluted and resuspended with 2, 4, 8 and 16 ml of DMEM/F12 addedat two-minute intervals. The cells are then pelleted and the supernatantaspirated. Two ml of DMEM/F12 supplemented with 15% FCS is carefullyadded to avoid resuspension of the pellet and then incubated for onehour at 37° C.

At the end of the 1 hour incubation, the suspension is diluted to afinal concentration of 1×10⁶ cells/ml. This suspension is then pouredinto tissue culture flasks and incubated overnight. The next day anequal volume of culture media supplemented with 2X HAT (hypoxanthine,aminopterin, thymidine) components are plated out into 96-well plateswith 200 μl/well with 1×10⁵ spleen cells/well. Plated cells are fedevery 4-5 days after the fusion by aspiration of half the media from thewells and addition of fresh 1×HAT media. The wells are scored for growthafter 10-14 days, and growing wells are tested for presence of secretedantibody by screening the culture supernatants by either an ELISA orWestern blotting. Wells that prove positive on assay are expanded forgrowth into 24-well culture dishes in culture media with HT supplements(no aminopterin) and re-tested. Those proving positive on re-testing areexpanded further into larger tissue culture vessels and then clonedtwice by limiting dilution.

A cell line (A4.85) that arose from a single mouse spleen cell wasisolated. A4.85 produces a monoclonal antibody (Mab) that reacts withseveral protein species (70 kilodaltons to several hundred kilodaltonsin mass) on a Western blot of FAM20 outer membranes, each of whosesynthesis is repressed by the presence of iron in the bacterial growthmedium.

Example 2A

Isolation of Genomic Clones

A. Library Construction

A library of Neisseria meningitidis strain FAM20 chromosomal DNA isconstructed in the bacteriophage vector lambda-gt11 as follows. FAM20chromosomal DNA is isolated by standard methods (Maniatis et al, 1982).The DNA is sheared by sonication to fragment sizes of approximately300-1000 bp. Synthetic EcoRI linkers are ligated to the ends of thesemolecules, followed by cleavage with EcoRI restriction endonuclease togenerate EcoRI restriction sites at the end of each molecule. Theresulting fragments are ligated with EcoRI-cleaved lambda-gt11 DNA(Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1982)). The ligatedDNA is packaged into lambda phage heads using lambda packaging extracts(Promega Corp., Madison, Wis.), according to manufacturer'sinstructions.

B. Library Screening and Isolation of DNA

The library created above is screened with the A4.85 Mab to detectclones that express the epitope recognized by A4.85. 500,000 recombinantplaques from the lambda-gt11 expression library are screened by themethod of Maniatis et al (1982). A pure clone reacting with the A4.85Mab is isolated by re-plating and screening the reactive plaque twice.The meningococcal insert DNA from the pure lambda clone (lambda 4.85) isamplified by the polymerase chain reaction (PCR) technique using a kitfrom Perkin-Elmer/Cetus. The PCR-amplified DNA is cloned into thesequencing vector M13mp19 (Maniatis et al, 1982) and the DNA sequencedetermined by the dideoxy chain termination method of Sanger et al(Proc. Natl. Acad. Sci. USA 74, 5463-5467 (19877)) using the Sequenasekit (Stratagene, La Jolla, Calif.).

The cloned meningococcal DNA is labelled with ³² P by the random primedmethod with a kit from Boehringer-Mannheim (Indianapolis, Ind.) and isused in Southern hybridizations (Maniatis et al, 1982) to identify DNArestriction fragments in the FAM20 chromosome adjacent to the DNA clonedin lambda 4.85. Chromosomal Sau3A I fragments of approximately 560 and1600 bp hybridize to the cloned meningococcal DNA. FAM20 DNA is cleavedwith Sau3A I and fractionated on a preparative agarose gel. Two sizefractions are isolated, one of 400-700 bp and one of 1400-1800 bp.

The 560 bp Sau3A I fragment is cloned by ligating the 400-700 bpfraction of FAM20-Sau3A I fragments with BamHI-cleaved plasmid PBR322(Maniatis et al, 1982). The desired clones of the 560 bp fragment areidentified by hybridization of bacterial colonies containing recombinantplasmids with ³² P-labelled lambda 4.85 insert DNA (Maniatis et al,1982). Plasmid DNA (pUNCH201) from a pure colony hybridizing with theDNA probe is prepared and its sequence determined using Sequenase asmodified for use in double-strand sequencing (Kraft et al, BioTechniques6, 544 (1988)). Southern hybridization is used to verify that the clonedfragment is representative of the fragment intact in the FAM20 genome.

To clone the 1600 bp fragment, the ends of the 1400-1800 bp fraction ofFAM20-Sau3A I fragments are made blunt by reaction with Klenow enzymeand DNA nucleotides. Synthetic EcoRI linkers are added to thesemolecules, followed by ligation with EcoRI-cleaved,alkaline-phosphatase-treated lambda ZAP DNA (Stratagene, LaJolla,Calif.) in accordance with technical information supplied with thelambda ZAP kit. Ligated DNA is packaged into lambda heads usingPackagene lambda packaging extracts (Promega). The library of 1400-1800bp FAM20-Sau3A I fragments is screened with a ³² P-labelledoligonucleotide (SAT1), which is synthesized to correspond to DNAsequences at one end of the lambda 4.85 insert (5' GCCATTGCCACTGTAGATA3'). A lambda ZAP plaque hybridizing with the SAT1 oligonucleotide ispurified as above. The interior portion of this lambda ZAP clone (lambdaZAP202) is "excised" by the addition of helper bacteriophage. Theexcision results in a multicopy plasmid (pUNCH202) containing the clonedmeningococcal insert. Southern hybridization is used to verify that thecloned fragment is representative of the fragment intact in the FAM20genome. The sequence of the cloned DNA fragment is determined bydouble-strand sequencing as described above.

To obtain the entire DNA sequence of the gene for an antigenic ironrepressible protein (frpA) from N. meningitidis related to the hemolysinfamily of toxins, two additional clones, pUNCH206 and pUNCH210, areisolated. These clones contain fragments flanking pUNCH201 and representthe entirety of the frpA gene.

To isolate pUNCH206, a 2.7 kilobase (kb) EcoRI to HincII fragmentoverlapping pUNCH201 is cloned as follows. FAM20 chromosomal DNA isdigested to completion with HincII. EcoRI linkers are ligated to theends of these fragments, which are then ligated to EcoRI-digested lambdaZAP. The desired clone is identified by hybridization with ³² P-labelledpUNCH201 insert. The interior portion of this clone is "excised" by theaddition of helper bacteriophage, and is designated pUNCH206. ThepUNCH206 insert contains the 3' end of the frpA gene.

To obtain pUNCH210, a 4.3 kb SpeI to ClaI fragment overlapping thepUNCH206 insert is cloned as follows. FAM20 DNA is digested with SpeIand NheI, the fragments are separated by pulsed-field gelelectrophoresis using a CHEF apparatus and are transferred to a BA-S85membrane. (Schleicher & Schuell, Keene, N.H.).

The fragment of interest is identified by hybridization. This fragmentis electrophoresed from the gel onto DE81 DEAE-cellulose paper andpurified. This DNA is digested with ClaI and the ends made blunt bytreatment with Klenow enzyme and DNA nucleotides. This is ligated withlambda ZAP which had been digested with EcoRI, then treated with Klenowfragment. The desired clone is identified by hybridization with ³²P-labelled pUNCH210 insert. The interior portion of this clone is"excised" by the addition of helper bacteriophage, and is designatedpUNCH210. The pUNCH210 insert contains the 5' end of the frpA gene.

The adjoined sequence of pUNCH210 and pUNCH206 reveals the presence ofan open reading frame that contains the entirety of the cloned DNA. TheDNA sequence is included in FIGS. 2A-G. See Seq. ID No. 1. The aminoacid sequence predicted by the sequence contains 1115 amino acids. Theputative amino acid sequence is shown as FIGS. 3A-E. See Seq. ID No. 2.The relationship to one another of the above-described restrictionfragments is shown in FIG. 1.

As determined by FASTA sequence comparison searches (see above), boththe DNA and the deduced polypeptide sequence from this region have ahigh degree of similarity with a family of hemolysin bacterial toxins.For example, the DNA sequence shown in FIGS. 2A-G (Seq. ID No. 1.)exhibits 54% identity with the cya gene (adenylate cyclase) from B.pertussis; 60% identity with the hlyA, hlyB, hlyC and hlyD gene from E.coli (hemolysin); 65% identity with hlyA, hlyB and hlyC gene (hemolysin)from E. coli; 56% identity with the leukotoxin gene from A.actinomycetemcomitans; 56% identity with the hemolysin gene from A.pleuropneumoniae; 60% identity with the leukotoxin gene from P.haemolytica; 62% identity with the A1 leukotoxin gene from P.Haemolytica; and 57% identity with protease B gene of E. chrysanthemi.

The amino acid sequence predicted from the DNA sequence exhibited25%-28% identity with leukotoxin, 22%-28% identity with hemolysin; and30% identity with adenylate cyclase.

Meningococcal strain FAM20 contains at least two copies of DNA thatencode the polypeptides of the invention. This can be demonstrated bydigesting genomic DNA with the infrequent cutters BglII, SpeI, NheI, andcombinations of NheI and SpeI. Southern blots of the digested DNAseparated by pulse field gradient electrophoresis reveal two major bandsthat hybridize under stringent conditions to gene probes containingfragments of the sequence of the gene that encodes the polypeptide ofthe invention.

The remainder of the gene encoding the iron-regulated polypeptide of theinvention is isolated in a manner similar to that described above forisolating pUNCH201 and pUNCH202. DNA restriction fragments eitherflanking the ends of the region already cloned or containing the entireregion are identified by Southern hybridization using oligonucleotideprobes derived from previously determined DNA sequence. These fragmentsare cloned into either plasmid or bacteriophage vectors as describedabove for pUNCH201 and pUNCH202. The DNA sequence of newly clonedfragments is determined as above, and reveals when either end of thegene is reached. If the gene is isolated on a single DNA fragment, it isexpressed in an in vitro assay to verify that the protein that isencoded by this gene reacts with the A4.85 Mab. If the gene is notcloned intact on a single DNA fragment, it is reconstructed throughstandard molecular biology techniques to yield the intact gene(Carbonetti, Proc. Natl. Acad. Sci. USA 84, 9084 (1987)).

For example, DNA fragments from one of the two copies of the structuralgenes coding for the polypeptide of the invention were purified fromagarose gels, cloned and sequenced. FIGS. 2A-G includes the DNAsequence. See Seq. ID No. 1.

Example 2B

Western Blot and Molecular Weight

The full length polypeptide obtained from meningococcal strain FAM20exhibits a molecular weight of 230-250 kD when subjected to Western blotanalysis. Western blots may be carried out as follows:

Iron-starved whole cells of FAM20 are prepared in accordance with themethod of West and Sparling, J. Bacteriol. 169, 3414-3421 (1987). Thecells are washed in ice-cold Davis Minimal Medium A (Lederberg, Methodsin Med. Res., 3:5 (1950)), immediately cooled on ice, and ruptured in aFrench pressure cell at 0° C. and 20,000 psi. The resulting mixture iscentrifuged for 10 minutes at 20,000 G, and the pellet solubilized inboiling SDS. The solubilized membrane proteins are separated by standard7.5% SDS-PAGE in Laemli buffer, which was described by Laemli in Nature227, 680-685 (1970). The proteins are transferred (16 hours, 80 μA) ontoOptibind nitrocellulose membranes (available from Schleicher & Schuell).The membranes are blocked for 1 hour in 5% BSA in TBS (20 mM Tris, 500Mm NaCl, pH 7.5); rinsed for 5 minutes in TBS; incubated for 1 hour with1:2 dilution of monoclonal antibody A4.85 (see above) in 5% BSA; washedtwice for 5 minutes in TBS and 0.05% Tween 20; incubated for 1 hour in asecondary antibody (goat anti-mouse lgG alkaline phosphatase conjugate)diluted in 5% BSA, available from BioRad (dilution=1:3000) or Sigma(dilution=1:1000); washed twice for 5 minutes in TBS/Tween; washed againfor 5 minutes in TBS; and developed with an alkaline phosphatasesubstrate comprising 45 μl Nitro Blue Tetrazolin, available from Sigma(75 mg/ml); 35 μl 5-bromo-4-chloro-3-indolylphosphate, p-tolnidine salt(50 mg/ml) in 10 ml of carbonate buffer, pH 9.8 (0.1 M NaHCO₃ ; 1 mMMgcl₂).

Example 3

Assay for Antibody in Sample

A standard ELISA protocol is used to screen for the presence ofantibodies against the polypeptide in proteins. Briefly, 96 wellmicrotiter plates are coated with the antigen at concentrations varyingfrom 50-100 ng per well in a high pH (9.6) carbonate buffer. The platesare incubated overnight at 9° C. and blocked with 10% normal goat serumfor one hour at 37° C. Patient sera is added and titered to determinethe endpoint. Control positive and negative sera is added at the sametime to quantitate the amount of relevant antibody present in theunknown samples. After a 2-3 hour incubation at 37° C., samples areprobed with goat anti-human Ig conjugated to horseradish peroxidase.Positive samples are determined by using TMB.

The invention as claimed is enabled in accordance with the specificationand readily available references and starting materials. Nevertheless,the following cell lines have been deposited in the American TypeCulture Collection, Bethesda, Md. on Jul. 12, 1990 in order tofacilitate the making and using of the invention:

Meningococcal cell line FAM18 (Accession Number 55071)

Meningococcal cell line FAM20 (Accession Number 55072)

Hybridoma cell line A4.85 (Accession Number HB 10504) In addition, thefollowing brochures containing useful protocols and information areavailable in the file history of this specification.

"Predigested Lambda Zap/Eco RI Cloning Kit Instruction Manual,"Stratagene, La Jolla, Calif. (Nov. 20, 1987);

"Gigapack Plus" (for packaging recombinant lambda phage), Stratagene, LaJolla, Calif. (Apr. 25, 1988); and

"picoBlue Immunoscreening Kit" Instruction Manual," Stratagene, LaJolla, Calif. (May 19, 1989).

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 2                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3758 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -      (v) FRAGMENT TYPE: N-terminal                                          -     (vi) ORIGINAL SOURCE:                                                   #meningitidis ORGANISM: Neisseria                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 298..3645                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: mat.sub.-- - #peptide                                           (B) LOCATION: 298..3642                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - TATATGTCTT TATTTGAATA TATCTTACGA TGGGGAAATA TTTATATATT TT - #ATAATAAA         60                                                                          - TTTTACTCAT TTGCTAATAT GTCATGGAAT ATTACTTGTA TTTTGTAGAA TT - #TTTCCATA        120                                                                          - TGAAAATATT CCATTTACTA TTTTTCTGAA CTTTATTAGT TTATTTTTAA TA - #TTTTTACC        180                                                                          - TCTTATATTT ACCATAAGAG AGCTAATTGA TTCATATTAT ATTGAGTCGA TA - #ATTAATTT        240                                                                          - ATTCTTAATT TTAATTCCTC ACGTTATTTT TTTAATTTAC TTGAAAGGAA AG - #CAGAT           297                                                                          - ATG ACA TCT GCA AAT TTT AAT ATT AAC GGT TT - #T GGA GAT GTG AAA TTA          345                                                                          Met Thr Ser Ala Asn Phe Asn Ile Asn Gly Ph - #e Gly Asp Val Lys Leu           #                 15                                                          - ACA CCC TAT TCA CCA CTC TTG GGA TAT AAA GC - #T TGG GAT TCA TTT ATT          393                                                                          Thr Pro Tyr Ser Pro Leu Leu Gly Tyr Lys Al - #a Trp Asp Ser Phe Ile           #             30                                                              - GGT TCT ATT CAA TCC TTA TCT GAT TTA ATC TA - #T AAT GTG GAT AAC AAT          441                                                                          Gly Ser Ile Gln Ser Leu Ser Asp Leu Ile Ty - #r Asn Val Asp Asn Asn           #         45                                                                  - AGA AAT AAA ATG GAA ATT ACT GTT AAT AAT GC - #T ATC CAA GCT GCA GAT          489                                                                          Arg Asn Lys Met Glu Ile Thr Val Asn Asn Al - #a Ile Gln Ala Ala Asp           #     60                                                                      - AGC TTT TTA AGC AGT ATT GGA AGA GAT AAC AA - #A ATA ACA AAT ACT GCT          537                                                                          Ser Phe Leu Ser Ser Ile Gly Arg Asp Asn Ly - #s Ile Thr Asn Thr Ala           # 80                                                                          - TCT TTA CTT GCA TCC CTC GAT AAC ATT TTT TT - #A AAT TTA AGA AAT GTA          585                                                                          Ser Leu Leu Ala Ser Leu Asp Asn Ile Phe Le - #u Asn Leu Arg Asn Val           #                 95                                                          - TCT CGA GAT ATA CGA GAA ACA GGA AAA TTT AA - #A CCT AAT GAT ATT CAA          633                                                                          Ser Arg Asp Ile Arg Glu Thr Gly Lys Phe Ly - #s Pro Asn Asp Ile Gln           #           110                                                               - CAA GCA ATT GGT GAT ATA TTC ATT GCT GCT GG - #T GAT GGA TTA CAA TAT          681                                                                          Gln Ala Ile Gly Asp Ile Phe Ile Ala Ala Gl - #y Asp Gly Leu Gln Tyr           #       125                                                                   - ATA AAA CAA CAA ACA GAG GCG ATG GCT CAA AG - #C AAA TTC TTA CCA ACT          729                                                                          Ile Lys Gln Gln Thr Glu Ala Met Ala Gln Se - #r Lys Phe Leu Pro Thr           #   140                                                                       - AAA TTA AAA ACT GGT TTA AAT GAT GTC CTT AA - #T TCT AGA ATG CTA AAA          777                                                                          Lys Leu Lys Thr Gly Leu Asn Asp Val Leu As - #n Ser Arg Met Leu Lys           145                 1 - #50                 1 - #55                 1 -       #60                                                                           - TCC TCT ACT GTT TTA CAG CAT GAA TTG AAT TA - #T TTG GGA TTT AAA ATA          825                                                                          Ser Ser Thr Val Leu Gln His Glu Leu Asn Ty - #r Leu Gly Phe Lys Ile           #               175                                                           - AAG GAT TAT GGA AAC GAG AGG CTT GGC GAA TC - #T ATA ATG AAT ATA GAT          873                                                                          Lys Asp Tyr Gly Asn Glu Arg Leu Gly Glu Se - #r Ile Met Asn Ile Asp           #           190                                                               - GAT TTT ACA CCA AGT AAG ATA GCA AAC TTT TT - #T GCG GAT CCT GAT ACA          921                                                                          Asp Phe Thr Pro Ser Lys Ile Ala Asn Phe Ph - #e Ala Asp Pro Asp Thr           #       205                                                                   - TAC AGC AAT GTA TTA GAA GAA GTA TCT AGG TT - #T ATA TAT TCC TTA GTT          969                                                                          Tyr Ser Asn Val Leu Glu Glu Val Ser Arg Ph - #e Ile Tyr Ser Leu Val           #   220                                                                       - CCT GAT GAT GCA AAC CCT TGG AAA GGG GGC GA - #A GAT TAT ATT GGA CGA         1017                                                                          Pro Asp Asp Ala Asn Pro Trp Lys Gly Gly Gl - #u Asp Tyr Ile Gly Arg           225                 2 - #30                 2 - #35                 2 -       #40                                                                           - GGG ATA AGT GAA TGG GGA GAG TTA CTG GAA AA - #A TGG TAT AAA CAA GAT         1065                                                                          Gly Ile Ser Glu Trp Gly Glu Leu Leu Glu Ly - #s Trp Tyr Lys Gln Asp           #               255                                                           - TTT CTC CCT TAT CTT GAA AAA GAA TGG GAC CA - #A TTT CCG AAA TTT GAA         1113                                                                          Phe Leu Pro Tyr Leu Glu Lys Glu Trp Asp Gl - #n Phe Pro Lys Phe Glu           #           270                                                               - GAT TGG CTG CCT GAA TTC CCT GAA TGG GCA AG - #A GAG TGG TTG AAA TTA         1161                                                                          Asp Trp Leu Pro Glu Phe Pro Glu Trp Ala Ar - #g Glu Trp Leu Lys Leu           #       285                                                                   - GAT CCC AAA CGT TCA GGC AAA TAT CAT GTC TA - #C GAC CCC CTC GCC CTA         1209                                                                          Asp Pro Lys Arg Ser Gly Lys Tyr His Val Ty - #r Asp Pro Leu Ala Leu           #   300                                                                       - GAT CTA GAC GGC GAC GGT ATA GAA ACC GTT GC - #T GCC AAA GGC TTT GCA         1257                                                                          Asp Leu Asp Gly Asp Gly Ile Glu Thr Val Al - #a Ala Lys Gly Phe Ala           305                 3 - #10                 3 - #15                 3 -       #20                                                                           - GGT GCA TTG TTC GAC CAC CGC AAT CAA GGC AT - #C CGC ACC GCC ACC GGT         1305                                                                          Gly Ala Leu Phe Asp His Arg Asn Gln Gly Il - #e Arg Thr Ala Thr Gly           #               335                                                           - TGG GTT TCT GCC GAT GAC GGT TTA CTC GTC CG - #C GAT TTG AAC GGC AAC         1353                                                                          Trp Val Ser Ala Asp Asp Gly Leu Leu Val Ar - #g Asp Leu Asn Gly Asn           #           350                                                               - GGC ATC ATC GAC AAC GGC GCG GAA CTC TTC GG - #C GAC AAC ACC AAA CTG         1401                                                                          Gly Ile Ile Asp Asn Gly Ala Glu Leu Phe Gl - #y Asp Asn Thr Lys Leu           #       365                                                                   - GCA GAC GGT TCT TTT GCC AAA CAC GGC TAT GC - #A GCT TTG GCC GAA TTG         1449                                                                          Ala Asp Gly Ser Phe Ala Lys His Gly Tyr Al - #a Ala Leu Ala Glu Leu           #   380                                                                       - GAT TCA AAC GGC GAC AAC ATC ATC AAC GCG GC - #A GAC GCC GCA TTC CAA         1497                                                                          Asp Ser Asn Gly Asp Asn Ile Ile Asn Ala Al - #a Asp Ala Ala Phe Gln           385                 3 - #90                 3 - #95                 4 -       #00                                                                           - ACC CTG CGT GTA TGG CAG GAT CTC AAC CAG GA - #C GGC ATT TCC CAA GCT         1545                                                                          Thr Leu Arg Val Trp Gln Asp Leu Asn Gln As - #p Gly Ile Ser Gln Ala           #               415                                                           - AAT GAA TTG CGT ACC CTT GAA GAA TTG GGT AT - #C CAA TCT TTG GAT CTC         1593                                                                          Asn Glu Leu Arg Thr Leu Glu Glu Leu Gly Il - #e Gln Ser Leu Asp Leu           #           430                                                               - GCC TAT AAA GAT GTA AAT AAA AAT CTC GGT AA - #C GGT AAC ACT TTG GCT         1641                                                                          Ala Tyr Lys Asp Val Asn Lys Asn Leu Gly As - #n Gly Asn Thr Leu Ala           #       445                                                                   - CAG CAA GGC AGC TAT ACC AAA ACA GAC GGT AC - #A ACC GCA AAA ATG GGG         1689                                                                          Gln Gln Gly Ser Tyr Thr Lys Thr Asp Gly Th - #r Thr Ala Lys Met Gly           #   460                                                                       - GAT TTA CTT TTA GCA GCC GAC AAT CTG CAC AG - #C CGC TTC AAA GAC AAA         1737                                                                          Asp Leu Leu Leu Ala Ala Asp Asn Leu His Se - #r Arg Phe Lys Asp Lys           465                 4 - #70                 4 - #75                 4 -       #80                                                                           - GTG GAA CTC ACT GCC GAA CAG GCA AAA GCC GC - #C AAT CTT GCG GGC ATC         1785                                                                          Val Glu Leu Thr Ala Glu Gln Ala Lys Ala Al - #a Asn Leu Ala Gly Ile           #               495                                                           - GGC CGT CTG CGC GAT TTG CGC GAA GCT GCC GC - #A TTG TCC GGC GAT TTG         1833                                                                          Gly Arg Leu Arg Asp Leu Arg Glu Ala Ala Al - #a Leu Ser Gly Asp Leu           #           510                                                               - GCC AAT ATG CTG AAA GCT TAT TCT GCC GCC GA - #A ACT AAA GAA GCA CAG         1881                                                                          Ala Asn Met Leu Lys Ala Tyr Ser Ala Ala Gl - #u Thr Lys Glu Ala Gln           #       525                                                                   - TTG GCA TTG TTA GAT AAT TTG ATT CAC AAA TG - #G GCG GAA ACC GAT TCG         1929                                                                          Leu Ala Leu Leu Asp Asn Leu Ile His Lys Tr - #p Ala Glu Thr Asp Ser           #   540                                                                       - AAC TGG GGC AAA AAA TCG CCA ATG CGA CTT TC - #A ACC GAT TGG ACG CAA         1977                                                                          Asn Trp Gly Lys Lys Ser Pro Met Arg Leu Se - #r Thr Asp Trp Thr Gln           545                 5 - #50                 5 - #55                 5 -       #60                                                                           - ACG GCT AAT GAA GGT ATT GCA CTG ACA CCA TC - #C CAA GTA GCA CAA CTA         2025                                                                          Thr Ala Asn Glu Gly Ile Ala Leu Thr Pro Se - #r Gln Val Ala Gln Leu           #               575                                                           - AAA AAG AAC GCT TTA GTT TCC CTT TCT GAT AA - #A GCT AAA GCA GCT ATT         2073                                                                          Lys Lys Asn Ala Leu Val Ser Leu Ser Asp Ly - #s Ala Lys Ala Ala Ile           #           590                                                               - GAC GCC GCC CGC GAC CGC ATT GCC GTG CTT GA - #T GCC TAC ACG GGG CAG         2121                                                                          Asp Ala Ala Arg Asp Arg Ile Ala Val Leu As - #p Ala Tyr Thr Gly Gln           #       605                                                                   - GAT TCC AGC ACA CTC TAT TAC ATG AGC GAA GA - #A GAC GCG CTT AAT ATC         2169                                                                          Asp Ser Ser Thr Leu Tyr Tyr Met Ser Glu Gl - #u Asp Ala Leu Asn Ile           #   620                                                                       - GTC AAA GTA ACC AAC GAT ACA TAC GAC CAT CT - #C GCC AAA AAC ATC TAC         2217                                                                          Val Lys Val Thr Asn Asp Thr Tyr Asp His Le - #u Ala Lys Asn Ile Tyr           625                 6 - #30                 6 - #35                 6 -       #40                                                                           - CAA AAC CTG TTG TTC CAA ACC CGT TTG CAG CC - #A TAT TTG AAT CAA ATC         2265                                                                          Gln Asn Leu Leu Phe Gln Thr Arg Leu Gln Pr - #o Tyr Leu Asn Gln Ile           #               655                                                           - AGT TTC AAA ATG GAA AAT GAT ACG TTC ACT TT - #G GAT TTT AGT GGT CTT         2313                                                                          Ser Phe Lys Met Glu Asn Asp Thr Phe Thr Le - #u Asp Phe Ser Gly Leu           #           670                                                               - GTT CAA GCA TTT AAC CAT GTC AAA GAA ACT AA - #T CCG CAA AAA GCT TTT         2361                                                                          Val Gln Ala Phe Asn His Val Lys Glu Thr As - #n Pro Gln Lys Ala Phe           #       685                                                                   - GTG GAT TTG GCC GAG ATG CTT GCA TAT GGC GA - #A CTT CGT TCT TGG TAT         2409                                                                          Val Asp Leu Ala Glu Met Leu Ala Tyr Gly Gl - #u Leu Arg Ser Trp Tyr           #   700                                                                       - GAA GGC CGA AGA CTA ATG GCC GAT TAT GTG GA - #G GAG GCA AAA AAA GCA         2457                                                                          Glu Gly Arg Arg Leu Met Ala Asp Tyr Val Gl - #u Glu Ala Lys Lys Ala           705                 7 - #10                 7 - #15                 7 -       #20                                                                           - GGT AAA TTT GAA GAT TAC CAG AAA GTG TTG GG - #T CAG GAG ACC GTT GCA         2505                                                                          Gly Lys Phe Glu Asp Tyr Gln Lys Val Leu Gl - #y Gln Glu Thr Val Ala           #               735                                                           - TTA TTA GCT AAA ACA TCG GGT ACG CAA GCA GA - #T GAT ATC CTG CAA AAT         2553                                                                          Leu Leu Ala Lys Thr Ser Gly Thr Gln Ala As - #p Asp Ile Leu Gln Asn           #           750                                                               - GTA GGC TTT GGT CAT AAT AAA AAT GTT TCT TT - #A TAT GGT AAT GAC GGC         2601                                                                          Val Gly Phe Gly His Asn Lys Asn Val Ser Le - #u Tyr Gly Asn Asp Gly           #       765                                                                   - AAC GAC ACT CTA ATC GGC GGT GCA GGC AAT GA - #T TAC TTG GAG GGC GGC         2649                                                                          Asn Asp Thr Leu Ile Gly Gly Ala Gly Asn As - #p Tyr Leu Glu Gly Gly           #   780                                                                       - AGC GGT TCG GAT ACT TAT GTC TTC GGC AAA GG - #C TTC GGT CAG GAT ACG         2697                                                                          Ser Gly Ser Asp Thr Tyr Val Phe Gly Lys Gl - #y Phe Gly Gln Asp Thr           785                 7 - #90                 7 - #95                 8 -       #00                                                                           - GTC TAT AAT TAC GAC TAC GCT ACC GGA CGC AA - #A GAC ATC ATC CGC TTT         2745                                                                          Val Tyr Asn Tyr Asp Tyr Ala Thr Gly Arg Ly - #s Asp Ile Ile Arg Phe           #               815                                                           - ACC GAC GGT ATT ACA GCC GAT ATG CTG ACT TT - #T ACC CGA GAG GGC AAC         2793                                                                          Thr Asp Gly Ile Thr Ala Asp Met Leu Thr Ph - #e Thr Arg Glu Gly Asn           #           830                                                               - CAT CTT CTT ATC AAG GCA AAA GAC GAC AGT GG - #A CAA GTG ACT GTT CAG         2841                                                                          His Leu Leu Ile Lys Ala Lys Asp Asp Ser Gl - #y Gln Val Thr Val Gln           #       845                                                                   - TCC TAT TTC CAG AAC GAT GGC TCA GGT GCT TA - #C CGT ATC GAT GAG ATT         2889                                                                          Ser Tyr Phe Gln Asn Asp Gly Ser Gly Ala Ty - #r Arg Ile Asp Glu Ile           #   860                                                                       - CAT TTC GAT AAC GGC AAA GTA CTG GAT GTT GC - #C ACT GTC AAA GAA CTG         2937                                                                          His Phe Asp Asn Gly Lys Val Leu Asp Val Al - #a Thr Val Lys Glu Leu           865                 8 - #70                 8 - #75                 8 -       #80                                                                           - GTA CAG CAA TCC ACC GAC GGT TCG GAC AGA TT - #G TAT GCC TAC CAA TCC         2985                                                                          Val Gln Gln Ser Thr Asp Gly Ser Asp Arg Le - #u Tyr Ala Tyr Gln Ser           #               895                                                           - GGA AGT ACC TTA AAT GGC GGA TTG GGC GAT GA - #C TAT CTG TAC GGT GCC         3033                                                                          Gly Ser Thr Leu Asn Gly Gly Leu Gly Asp As - #p Tyr Leu Tyr Gly Ala           #           910                                                               - GAC GGG AAT GAC CTG CTG AAT GGT GAT GCA GG - #C AAC GAC AGT ATC TAC         3081                                                                          Asp Gly Asn Asp Leu Leu Asn Gly Asp Ala Gl - #y Asn Asp Ser Ile Tyr           #       925                                                                   - AGT GGC AAT GGC AAT GAT ACG CTC GAT GGA GG - #A GAA GGC AAC GAC GCC         3129                                                                          Ser Gly Asn Gly Asn Asp Thr Leu Asp Gly Gl - #y Glu Gly Asn Asp Ala           #   940                                                                       - CTG TAC GGC TAT AAT GGT AAC GAT GCA CTG AA - #T GGT GGC GAA GGC AAT         3177                                                                          Leu Tyr Gly Tyr Asn Gly Asn Asp Ala Leu As - #n Gly Gly Glu Gly Asn           945                 9 - #50                 9 - #55                 9 -       #60                                                                           - GAT CAT TTG AAC GGC GAA GAC GGT AAC GAC AC - #T CTA ATC GGC GGT GCA         3225                                                                          Asp His Leu Asn Gly Glu Asp Gly Asn Asp Th - #r Leu Ile Gly Gly Ala           #               975                                                           - GGC AAT GAT TAC TTG GAG GGC GGC AGC GGT TC - #G GAT ACT TAT GTC TTC         3273                                                                          Gly Asn Asp Tyr Leu Glu Gly Gly Ser Gly Se - #r Asp Thr Tyr Val Phe           #           990                                                               - GGC GAA GGC TTC GGT CAG GAT ACG GTC TAT AA - #T TAC CAT GTG GAT AAA         3321                                                                          Gly Glu Gly Phe Gly Gln Asp Thr Val Tyr As - #n Tyr His Val Asp Lys           #      10050                                                                  - AAC TCT GAC ACT ATG CAC TTT AAA GGA TTT AA - #A GCA GCA GAT GTT CAT         3369                                                                          Asn Ser Asp Thr Met His Phe Lys Gly Phe Ly - #s Ala Ala Asp Val His           #  10205                                                                      - TTT ATC CGT TCC GGA AGT GAT TTG GTG CTT AG - #C GCT TCT GAA CAA GAC         3417                                                                          Phe Ile Arg Ser Gly Ser Asp Leu Val Leu Se - #r Ala Ser Glu Gln Asp           #               10401030 - #                1035                              - AAC GTA CGT ATT TCC GGA TTC TTC TAT GGT GA - #A AAC CAT CGT GTA GAT         3465                                                                          Asn Val Arg Ile Ser Gly Phe Phe Tyr Gly Gl - #u Asn His Arg Val Asp           #              10550                                                          - ACA TTT GTC TTT GAT GAT GCA GCT ATC AGT AA - #T CCA GAT TTT GCC AAG         3513                                                                          Thr Phe Val Phe Asp Asp Ala Ala Ile Ser As - #n Pro Asp Phe Ala Lys           #          10705                                                              - TAT ATT AAT GCT GGC AAT AAT TTG GTA CAG TC - #T ATG TCT GTG TTC GGT         3561                                                                          Tyr Ile Asn Ala Gly Asn Asn Leu Val Gln Se - #r Met Ser Val Phe Gly           #      10850                                                                  - TCT AAT ACT GCT GCG ACA GGA GGA AAT GTG GA - #T GCC AAT ATA CAA TCC         3609                                                                          Ser Asn Thr Ala Ala Thr Gly Gly Asn Val As - #p Ala Asn Ile Gln Ser           #  11005                                                                      - GTA CAG CAG CCG TTA TTG GTA ACG CCA TCT GC - #A TAAGGAGCCT AATCACATTC       3662                                                                          Val Gln Gln Pro Leu Leu Val Thr Pro Ser Al - #a                               1105                1110 - #                1115                              - ATGGCTTAAA CTGAAAAACA GCAATCAAGT TTATTTTGAT TGCTGTTTTT CT - #TAATATTG       3722                                                                          #     3758         CTTT GCAAAAATAG TCTGTT                                     - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1115 amino                                                        (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Thr Ser Ala Asn Phe Asn Ile Asn Gly Ph - #e Gly Asp Val Lys Leu         #                 15                                                          - Thr Pro Tyr Ser Pro Leu Leu Gly Tyr Lys Al - #a Trp Asp Ser Phe Ile         #             30                                                              - Gly Ser Ile Gln Ser Leu Ser Asp Leu Ile Ty - #r Asn Val Asp Asn Asn         #         45                                                                  - Arg Asn Lys Met Glu Ile Thr Val Asn Asn Al - #a Ile Gln Ala Ala Asp         #     60                                                                      - Ser Phe Leu Ser Ser Ile Gly Arg Asp Asn Ly - #s Ile Thr Asn Thr Ala         # 80                                                                          - Ser Leu Leu Ala Ser Leu Asp Asn Ile Phe Le - #u Asn Leu Arg Asn Val         #                 95                                                          - Ser Arg Asp Ile Arg Glu Thr Gly Lys Phe Ly - #s Pro Asn Asp Ile Gln         #           110                                                               - Gln Ala Ile Gly Asp Ile Phe Ile Ala Ala Gl - #y Asp Gly Leu Gln Tyr         #       125                                                                   - Ile Lys Gln Gln Thr Glu Ala Met Ala Gln Se - #r Lys Phe Leu Pro Thr         #   140                                                                       - Lys Leu Lys Thr Gly Leu Asn Asp Val Leu As - #n Ser Arg Met Leu Lys         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ser Ser Thr Val Leu Gln His Glu Leu Asn Ty - #r Leu Gly Phe Lys Ile         #               175                                                           - Lys Asp Tyr Gly Asn Glu Arg Leu Gly Glu Se - #r Ile Met Asn Ile Asp         #           190                                                               - Asp Phe Thr Pro Ser Lys Ile Ala Asn Phe Ph - #e Ala Asp Pro Asp Thr         #       205                                                                   - Tyr Ser Asn Val Leu Glu Glu Val Ser Arg Ph - #e Ile Tyr Ser Leu Val         #   220                                                                       - Pro Asp Asp Ala Asn Pro Trp Lys Gly Gly Gl - #u Asp Tyr Ile Gly Arg         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Gly Ile Ser Glu Trp Gly Glu Leu Leu Glu Ly - #s Trp Tyr Lys Gln Asp         #               255                                                           - Phe Leu Pro Tyr Leu Glu Lys Glu Trp Asp Gl - #n Phe Pro Lys Phe Glu         #           270                                                               - Asp Trp Leu Pro Glu Phe Pro Glu Trp Ala Ar - #g Glu Trp Leu Lys Leu         #       285                                                                   - Asp Pro Lys Arg Ser Gly Lys Tyr His Val Ty - #r Asp Pro Leu Ala Leu         #   300                                                                       - Asp Leu Asp Gly Asp Gly Ile Glu Thr Val Al - #a Ala Lys Gly Phe Ala         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Gly Ala Leu Phe Asp His Arg Asn Gln Gly Il - #e Arg Thr Ala Thr Gly         #               335                                                           - Trp Val Ser Ala Asp Asp Gly Leu Leu Val Ar - #g Asp Leu Asn Gly Asn         #           350                                                               - Gly Ile Ile Asp Asn Gly Ala Glu Leu Phe Gl - #y Asp Asn Thr Lys Leu         #       365                                                                   - Ala Asp Gly Ser Phe Ala Lys His Gly Tyr Al - #a Ala Leu Ala Glu Leu         #   380                                                                       - Asp Ser Asn Gly Asp Asn Ile Ile Asn Ala Al - #a Asp Ala Ala Phe Gln         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Thr Leu Arg Val Trp Gln Asp Leu Asn Gln As - #p Gly Ile Ser Gln Ala         #               415                                                           - Asn Glu Leu Arg Thr Leu Glu Glu Leu Gly Il - #e Gln Ser Leu Asp Leu         #           430                                                               - Ala Tyr Lys Asp Val Asn Lys Asn Leu Gly As - #n Gly Asn Thr Leu Ala         #       445                                                                   - Gln Gln Gly Ser Tyr Thr Lys Thr Asp Gly Th - #r Thr Ala Lys Met Gly         #   460                                                                       - Asp Leu Leu Leu Ala Ala Asp Asn Leu His Se - #r Arg Phe Lys Asp Lys         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Val Glu Leu Thr Ala Glu Gln Ala Lys Ala Al - #a Asn Leu Ala Gly Ile         #               495                                                           - Gly Arg Leu Arg Asp Leu Arg Glu Ala Ala Al - #a Leu Ser Gly Asp Leu         #           510                                                               - Ala Asn Met Leu Lys Ala Tyr Ser Ala Ala Gl - #u Thr Lys Glu Ala Gln         #       525                                                                   - Leu Ala Leu Leu Asp Asn Leu Ile His Lys Tr - #p Ala Glu Thr Asp Ser         #   540                                                                       - Asn Trp Gly Lys Lys Ser Pro Met Arg Leu Se - #r Thr Asp Trp Thr Gln         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Thr Ala Asn Glu Gly Ile Ala Leu Thr Pro Se - #r Gln Val Ala Gln Leu         #               575                                                           - Lys Lys Asn Ala Leu Val Ser Leu Ser Asp Ly - #s Ala Lys Ala Ala Ile         #           590                                                               - Asp Ala Ala Arg Asp Arg Ile Ala Val Leu As - #p Ala Tyr Thr Gly Gln         #       605                                                                   - Asp Ser Ser Thr Leu Tyr Tyr Met Ser Glu Gl - #u Asp Ala Leu Asn Ile         #   620                                                                       - Val Lys Val Thr Asn Asp Thr Tyr Asp His Le - #u Ala Lys Asn Ile Tyr         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Gln Asn Leu Leu Phe Gln Thr Arg Leu Gln Pr - #o Tyr Leu Asn Gln Ile         #               655                                                           - Ser Phe Lys Met Glu Asn Asp Thr Phe Thr Le - #u Asp Phe Ser Gly Leu         #           670                                                               - Val Gln Ala Phe Asn His Val Lys Glu Thr As - #n Pro Gln Lys Ala Phe         #       685                                                                   - Val Asp Leu Ala Glu Met Leu Ala Tyr Gly Gl - #u Leu Arg Ser Trp Tyr         #   700                                                                       - Glu Gly Arg Arg Leu Met Ala Asp Tyr Val Gl - #u Glu Ala Lys Lys Ala         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Gly Lys Phe Glu Asp Tyr Gln Lys Val Leu Gl - #y Gln Glu Thr Val Ala         #               735                                                           - Leu Leu Ala Lys Thr Ser Gly Thr Gln Ala As - #p Asp Ile Leu Gln Asn         #           750                                                               - Val Gly Phe Gly His Asn Lys Asn Val Ser Le - #u Tyr Gly Asn Asp Gly         #       765                                                                   - Asn Asp Thr Leu Ile Gly Gly Ala Gly Asn As - #p Tyr Leu Glu Gly Gly         #   780                                                                       - Ser Gly Ser Asp Thr Tyr Val Phe Gly Lys Gl - #y Phe Gly Gln Asp Thr         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Val Tyr Asn Tyr Asp Tyr Ala Thr Gly Arg Ly - #s Asp Ile Ile Arg Phe         #               815                                                           - Thr Asp Gly Ile Thr Ala Asp Met Leu Thr Ph - #e Thr Arg Glu Gly Asn         #           830                                                               - His Leu Leu Ile Lys Ala Lys Asp Asp Ser Gl - #y Gln Val Thr Val Gln         #       845                                                                   - Ser Tyr Phe Gln Asn Asp Gly Ser Gly Ala Ty - #r Arg Ile Asp Glu Ile         #   860                                                                       - His Phe Asp Asn Gly Lys Val Leu Asp Val Al - #a Thr Val Lys Glu Leu         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Val Gln Gln Ser Thr Asp Gly Ser Asp Arg Le - #u Tyr Ala Tyr Gln Ser         #               895                                                           - Gly Ser Thr Leu Asn Gly Gly Leu Gly Asp As - #p Tyr Leu Tyr Gly Ala         #           910                                                               - Asp Gly Asn Asp Leu Leu Asn Gly Asp Ala Gl - #y Asn Asp Ser Ile Tyr         #       925                                                                   - Ser Gly Asn Gly Asn Asp Thr Leu Asp Gly Gl - #y Glu Gly Asn Asp Ala         #   940                                                                       - Leu Tyr Gly Tyr Asn Gly Asn Asp Ala Leu As - #n Gly Gly Glu Gly Asn         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Asp His Leu Asn Gly Glu Asp Gly Asn Asp Th - #r Leu Ile Gly Gly Ala         #               975                                                           - Gly Asn Asp Tyr Leu Glu Gly Gly Ser Gly Se - #r Asp Thr Tyr Val Phe         #           990                                                               - Gly Glu Gly Phe Gly Gln Asp Thr Val Tyr As - #n Tyr His Val Asp Lys         #      10050                                                                  - Asn Ser Asp Thr Met His Phe Lys Gly Phe Ly - #s Ala Ala Asp Val His         #  10205                                                                      - Phe Ile Arg Ser Gly Ser Asp Leu Val Leu Se - #r Ala Ser Glu Gln Asp         #               10401030 - #                1035                              - Asn Val Arg Ile Ser Gly Phe Phe Tyr Gly Gl - #u Asn His Arg Val Asp         #              10550                                                          - Thr Phe Val Phe Asp Asp Ala Ala Ile Ser As - #n Pro Asp Phe Ala Lys         #          10705                                                              - Tyr Ile Asn Ala Gly Asn Asn Leu Val Gln Se - #r Met Ser Val Phe Gly         #      10850                                                                  - Ser Asn Thr Ala Ala Thr Gly Gly Asn Val As - #p Ala Asn Ile Gln Ser         #  11005                                                                      - Val Gln Gln Pro Leu Leu Val Thr Pro Ser Al - #a                             1105                1110 - #                1115                              __________________________________________________________________________

What is claimed is:
 1. An essentially pure polypeptide comprising theamino acid sequence shown in SEQ ID No.
 2. 2. A method for detecting thepresence of a polypeptide according to claim 1 in a sample suspected ofcontaining the polypeptide, the method comprising the steps of:(a)contacting the sample with a labelled antibody that binds to thepolypeptide; and (b) detecting the presence of the label bound to thepolypeptide.
 3. A method for detecting the presence of an antibodyspecific for the polypeptide according to claim 1 in a sample suspectedof containing the antibody the method comprising the steps of:(a)immobilizing the polypeptide on a solid support; (b) contacting thesample with the immobilized polypeptide to bind any antibodies in thesample; and (c) detecting the presence of the bound antibody.